The long term goal of this project is to elucidate genetic pathways regulating early determination and development of the mesencephalon (midbrain) and metencephalon (cerebellum). Recent transplantation studies with chick embryos an genetic analysis in mice have indicated that formation of the mesencephalon and metencephalon (mes/met) require Wnt1 and En, that the isthmus acts as an organizer for the region with Fgf8 being its likely inducing factor, and that Gbx2 may act later in cerebellum development. An important question remaining is what are the roles of EN1, Fgf8 and Gbx2 in mes/met development. Specific aims: 1. To determine whether En1 can transform diencephalon cells into midbrain cells by expressing EN1 in the dorsal forebrain form the Wnt7b locus using a gene targeting approach (knock-in). 2. To confirm that in mouse Fgf8 can induce a midbrain development in the diencephalon and determine whether Fgf8 can induce cerebellum development in other regions of the brain. Fgf8 also will be ectopically expressed on En and Gbx2 mutant background to determine whether these genes are downstream targets of Fgf8 signaling. 3. O determine whether Gbx2 has multiple roles during mes/met development by making conditional Gbx2 mutants that lack Gbx2 function after embryonic day 8.5. 4. To determine whether Gbx2 can transform regions of the brain into a cerebellum by expressing it alone, or with En/Fgf8, in the dorsal forebrain and mes of transgenic mice. These studies should provide a new information on the genetic control of early mes/met development and in a broader context insight into general mechanisms of early mammalian brain development. The information gained using mouse as a model mammal should lead to the identification of genes causing human birth defects and suggest possible methods of treatment or prevention.